Mechanisms Involved in Flavin-linked Oxygen Metabolism

描述：(申请人提供)已知的乳酸菌 代表了氧气代谢中生物化学多样性的有力范例， 由于它们缺乏呼吸细胞色素和其他血红蛋白。取而代之的是 这些生物依赖于不寻常的黄素连接呼吸酶网络， 这种应用主要集中在详细的结构和力学研究上 这一群体的三个特别令人感兴趣的代表：(1)NADH 来自人类严重病原体的氧化酶(NOx；2NADH+O2至2NAD++2H2O) 化脓性链球菌，(2)NADH过氧化物酶(Npx；NADH+H2O2-to NAD++2H2O) 和(3)α-甘油磷酸酶(GlpO； α-甘油磷酸盐+O2-合成磷酸二羟丙酮+双氧水) 链球菌属GlpO序列与该序列密切相关 膜相关α-甘油磷酸脱氢酶(GlpDS)；这些酶是 主要区别在于存在GlpO独有的50个残基插入， 这进一步在有限的蛋白质分解实验中显示出来 表示在软化过程中起关键作用的灵活表面区 还原。对GlpO结构的阐明应该有助于深入了解 以及相关GIPD的结构，并应更好地界定 甘油磷酸氧化过程中，GlpO表面形成环状。而结构上的 新的晶体大大加强了NOx和Npx之间的同源性 结构，只是最近才有第三个成员。 在这一小类NAD(P)H连接的过氧化氢/二硫化物还原酶中 FAD依赖的辅酶A-二硫键还原酶(CoADR；NADPH+ CoASSCoA到NADP++2CoASH)从人类严重病原体葡萄球菌 金星。CoADR很好地补充了这一新兴的黄素蛋白组， 利用单个活性中心半胱氨酸作为基本氧化还原中心的基础- 与NOx和Npx一起记录的半胱氨酸(Cys-SOH)，以及Cys-SSCoA CoADR中存在二硫化物。这是一个额外的长期目标 应用是对结构性、氧化还原和机械性的持续定义 NOx和Npx中Cys-SOH功能的参数，因为这些酶提供了 目前已知的蛋白质-SOH中心参与不同 对氧化还原信号和催化方面进行了分析。新的结构 可用于NOx和Npx也为继续分析 相对罕见的O2还原为2H2O的功能区别 由NOx和Npx的过氧化反应催化。可解读的 最近为CoADR计算的电子密度图现在使得有可能 对这种酶的原子结构进行建模和提炼，从而允许 与NOx和Npx的详细比较，并提供对 这种不寻常的二硫键表现出功能不对称的基础 还原酶。

DESCRIPTION: (provided by applicant) Lactic acid bacteria are known to represent strong paradigms for the biochemical diversity in oxygen metabolism, due to their lack of respiratory cytochromes and other hemeproteins. Instead these organisms rely on networks of unusual flavin-linked respiratory enzymes, and this application is focused on detailed structural and mechanistic studies of three particularly interesting representatives of this group: (1) NADH oxidase (Nox; 2NADH + O2 to 2NAD+ + 2H2O) from the severe human pathogen Streptococcus pyogenes, (2) NADH peroxidase (Npx; NADH + H2O2 - to NAD+ + 2H2O) from Enterococcus faecalis, and (3) alpha-glycerophosphate oxidase (GlpO; alpha-glycerophosphate + O2 - to dihydroxyacetone phosphate + H2O2) from Streptococcus sp. The GlpO sequence is closely related to those of the membrane-associated a-glycerophosphate dehydrogenases (GlpDs); the enzymes are distinguished primarily by the presence of a 50-residue insert unique to GlpO, which has furthermore been shown in limited proteolysis experiments to represent a flexible surface region that plays a crucial role in flavin reduction. Elucidation of the GlpO structure should provide insight into the structures of the related GIpDs as well and should better define the role of the GlpO surface loop during aglycerophosphate oxidation. While the structural homology between Nox and Npx has been reinforced dramatically with new crystal structures during the current project period, only recently has a third member of this small class of NAD(P)H-linked peroxide/disulfide reductases been identified - the FAD-dependent coenzyme A-disulfide reductase (CoADR; NADPH + CoASSCoA to NADP+ + 2CoASH) from the serious human pathogen Staphylococcus aureus. CoADR nicely complements this emerging group of flavoproteins which utilize a single active-site Cys as the basis for an essential redox center - the Cys-sulfenic acid (Cys-SOH) documented with Nox and Npx, and the Cys-SSCoA disulfide present in CoADR. An additional long-term objective of this application is the ongoing definition of the structural, redox, and mechanistic parameters for Cys-SOH function in Nox and Npx, as these enzymes provide the benchmarks by which protein-SOH centers now known to participate in diverse aspects of redox signaling and catalysis are analyzed. The new structures available for Nox and Npx also provide the basis for continuing analyses of the functional distinction between the relatively rare reduction of O2 to 2H2O catalyzed by Nox and the peroxidatic reaction of Npx. The interpretable electron density map recently calculated for CoADR now makes it possible to model and refine the atomic structure of this enzyme as well, thus allowing detailed comparisons with Nox and Npx and providing structural insights into the basis for the functional asymmetry exhibited by this unusual disulfide reductase.